Composite liquid cells

ABSTRACT

A sample handling method may include drawing an encapsulating liquid from an encapsulating-liquid input; discharging the drawn encapsulating liquid (a) onto a free surface of a carrier liquid in a carrier-liquid conduit comprising a stabilisation feature and (b) proximate to the stabilisation feature, the encapsulating liquid being immiscible with the carrier liquid, so that the discharged encapsulating liquid does not mix with the carrier liquid, floats on top of the carrier liquid, and is immobilised by the stabilisation feature; drawing a sample liquid from a sample-liquid input; and discharging the drawn sample liquid, the sample liquid being immiscible with the encapsulating liquid and with the carrier liquid, so that the sample liquid does not mix with the encapsulating liquid or with the carrier liquid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.Nos. 61/344,434, filed Jul. 22, 2010, 61/470,515, filed Apr. 1, 2011,and 61/470,520, filed Apr. 1, 2011, each of which is hereby incorporatedherein by reference.

BACKGROUND

Currently the processing of biochemistry samples has a number of keydrawbacks. These include the volume size—resulting in high reagentcosts; high consumable costs; and labour-intensive protocols andprocesses which are highly susceptible to cross-contamination. For thesereasons complete control and isolation of each individual sample withinthe biochemistry process cannot currently be ensured.

For a number of biochemistry process applications—sequence beadpreparation, pyrosequencing, nucleic acid ligation, and polymerase chainreaction—and not limited to these, the limitations of volume size,chemistry cost, labour cost, and the reaction efficiency are evident.

Sequence bead preparation is a process by which small beads are coatedin an application-specific chemistry. For example in DNA replication,the beads are coated initially with DNA primers in advance of theamplification process. Even for today's state-of-the-art sequencers arelatively high local concentration of the target molecule is requiredto sequence accurately. Current estimates for a typical protocolestimate that only 80% of the beads processed are sufficiently coated toensure accurate sequencing. Therefore to ensure a relatively highconcentration of the target sample a large number of beads must be usedfor statistical accuracy. Furthermore, the transferal of even coatedbeads from one well to another inevitably leads to losses of both thebeads and the suspended fluid. This is a result of dead volumes andinefficiencies inherent in today's pipetting and liquid handlingsystems. This biochemistry process is generally performed in 96 or 384static well plates with typical volumes ranging from 10 microlitres to200 microlitres.

Another biochemistry process, pyrosequencing, mixes a relatively highconcentration of nucleic acid with primer-coated beads. The nucleicacids attach and form a clonal colony on the beads. This is thenamplified using emulsion-based PCR. The sequencing machine contains alarge number of picolitre-volume wells that are large enough for asingle bead along with the relevant enzymes required for sequencing.Pyrosequencing uses the luciferase enzyme to generate light as read-out,and the sequencing machine takes a picture of the wells for every addednucleotide. One of the key difficulties in this process is the efficientcoating of the beads with primers. A percentage of beads using currenttechnologies are not properly coated with primer chemistry, resulting inpoorer reaction efficiencies. Using today's technologies to improve thecoating efficiencies of the beads would require an unsustainableincrease in reagent cost.

Within nucleic acid ligation similar biochemistry processing issuesarise. Nucleic acid ligation has become an important tool in modernmolecular biology research for generating recombinant nucleic acidsequences. For example, nucleic acid ligases are used with restrictionenzymes to insert nucleic acid fragments, often genes, into plasmids foruse in genetic engineering. Nucleic acid ligation is a relatively commontechnique in molecular biology wherein short strands of DNA may bejoined together by the action of an enzyme called ligase at a specifictemperature, commonly 16-25° C. depending on the protocol used. To joinmore than two sequences of short DNA strands together, for example inthe construction of a synthetic genetic sequence, it is impossible tocombine all the DNA strands and then perform the ligation. This wouldresult in random sequences in which the end of one strand would bejoined to the start of an incorrect strand. This incorrect sequence, ororientation, would not be desirable in a synthetically-constructed genewhere the order of the genetic code is crucial. To perform the techniquecorrectly pairwise combinations of neighbouring sequences must first beligatated to yield the correct orientation. These paired syntheticconstructs may then be ligated in the correct orientation to yield evenlonger synthetic constructs. The process involves a large and intricateamount of chemistry processing and manipulation. This can be quite alabour intensive process or if performed using today's liquid handlingand results in large consumable costs and suffers from the known deadvolume losses of the static well plates and pipette aspirations. Alsousing today's liquid handling technologies the mixing and control ofsmall volumes is limited by the ability to aspirate and manipulaterelatively small volumes. Typical volumes used in nucleic acid ligationare 10-200 microlitres with nucleic acid strand lengths between 50-200base pairs.

Polymerase Chain Reaction (PCR) has been used extensively to amplifytargeted DNA and cDNA for many applications in molecular biology. ThePCR technique amplifies a single or a few copies of a piece of DNA,generating thousands to billions of copies of a particular DNA sequence.Modem PCR instruments carry out the PCR process in reaction volumesranging from 10-200 micro-litres. One of the largest obstacles tocarrying out PCR in small volumes is the difficulty in manipulatingsmall volumes of the constituent reagents with manual pipettes. Thelarge volume size is a direct result of the poor capability of existingtechnologies to dispense and mix sub-nanolitre volumes. Furthermore, forthe next generation microfluidic technologies based on flowing systems,these are still limited by the starting volume dispensed versus theactual amount of sample required for the biochemistry process. Thesemicrofluidic systems are also limited during the biochemistry process toa defined protocol control of the samples. These systems typically relyon micro-scale fluid channel networks to transport and mixsub-microlitre volumes. Some of the major drawbacks of thesetechnologies are: the single use of the microfluidic cards—to preventcontamination—the lack of dynamic control of the each individualsample—transporting and mixing any individual sample at any point in thebiochemistry process—and the closed architecture of the system.

In particular, current methods of Digital Polymerase Chain Reaction(dPCR) are performed through the division of an initial sample intomultiple smaller volumes samples until one DNA template remains in eachsubvolume. Counting the number of positive subvolumes which contain DNA,the starting copy number in the original volume can be calculated.Typically, this involves multiple serial dilution steps to generate asample volume with statistically one DNA target per reaction volume.Statistically a subset of the total volume may be tested to determinethe initial copy number, allowing for a reduction in the total number ofPCR reactions. However for rare target detection a larger subset ofvolumes need to be tested to improve the statistical accuracy. Thisresults in a larger number of blank volumes and a larger testvolume—resulting in the use of more chemistry, time, instrumentation,sample handling, and processing steps.

Another method of dPCR is whereby an emulsion of the test volume isgenerated in an oil-based carrier. This method is an effort to reducethe number of instruments required and time required for a result.First, the target sample is diluted and emulsified into small enoughvolumes with a statistical distribution of less than one copy perdroplet, within the carrier oil. This larger volume can then be treatedas a single sample volume and processed using PCR protocols. Howeverthis method is generally limited to end point detection. Furtherinstrumentation is required in the form of a flow cytometer, therebybeing able to detect the target presence per droplet flowing past asensor. Flow cytometers are low speed; expensive; can require specificfluid mediums and only allow for endpoint detection. The limitations ofendpoint detection include the requirement of a post processing step;lower sensitivity; longer time to result; specificity and moreinstrumentation. A further challenge for emulsion based PCR methods isthe stability required and control of each droplet. Droplet merging orsplitting introduces further statistical errors into the processing.

Today's pipetting and liquid handling systems are unable to process 100%of the given starting volume. For pipettes both the liquid storagesystem—static well plates—and the mechanical actuation within the systemprevent complete aspiration of the sample. This loss or dead volume instatic plates can be accounted for by the surface wettingcharacteristics and the geometry, neither of which current technologiescan account for.

In flowing systems the collection of individual biological samplesduring or at the end of the biochemistry process is proving to be verychallenging for existing technologies. The typical continuous flowingsystems comprise of pumps and reservoirs which generally make the easyretrieval of critical fluids, particularly at the microscale,technically difficult. Also, within flowing systems initial priming ofthe system is time consuming, costly and if done incorrectly leads to acatastrophic failure of the test requiring a retest of the biologicalsample.

Another drawback to existing biochemistry processing is the inability toautomate the biochemistry process for nano-litre and sub-nano-litrevolumes. The transport, mixing or retrieval of each individual samplecannot be performed by existing automated technologies.

In more general chemistry processing, such as generic microchemistry,where the manipulation of small amounts of fluid is necessary, one canclearly see the limitations of current technology in the volume wastefluid remaining in the static well plates or within the system. This isa result of current technology's lack of capability to dispense andcontrol smaller volumes demanded by evermore sophisticated molecularbiology techniques, and the call for improved efficiencies.

The invention is therefore directed towards providing improved samplehandling to overcome at least some of the above problems.

SUMMARY

Devices, systems and methods for making and handling composite liquidcells are disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B schematically illustrate composite liquid cellgeneration using electrostatic forces.

FIGS. 2A and 2B schematically illustrate composite liquid cellgeneration using the hydrophobic effect.

FIGS. 3A and 3B schematically illustrate composite liquid cellgeneration using directional air control.

FIGS. 4A-4F schematically illustrate generation of a composite liquidcell using a control tube and variable flow direction;

FIG. 5 schematically illustrates control of a composite liquid cellusing electrostatic forces.

FIGS. 6A-6C schematically illustrate control of a composite liquid cellusing the hydrophobic effect.

FIG. 7 schematically illustrates control of a composite liquid cellusing directional air control.

FIG. 8 schematically illustrates a static control spur anchoring acomposite liquid cell.

FIGS. 9A and 9B schematically illustrate a transport mechanism forcontinuous flowing processing of biochemistry along a static hydrophobiccontrol surface using electrostatic forces.

FIGS. 10A-10F schematically illustrate multi-sample composite liquidcell generation of the invention using a control tube and a variableflow direction.

FIGS. 11A-11C schematically illustrate multi-sample composite liquidcell generation using electrostatic forces.

FIGS. 12A and 12B are photographs showing a multi-sample compositeliquid cell.

FIGS. 13A-13C schematically illustrate multi-sample composite liquidcell generation using surface tension.

FIGS. 14A and 14B schematically illustrate multi-sample composite liquidcell generation using mechanical agitation.

FIGS. 15A and 15B are photographs of an emulsion-based multi-samplecomposite liquid cell.

FIG. 16 is a photograph of a multi-sample composite liquid cell withmultiple internal sample targets.

FIGS. 17A and 17B schematically illustrate multi-sample composite cellgenerating using directional air control.

FIGS. 18A-18C schematically illustrate control of a multi-samplecomposite liquid cell using the hydrophobic effect.

FIGS. 19A and 19B schematically illustrate multi-sample composite celltransportation using a hydrophobic control surface with stabilisationfeatures.

FIG. 20 schematically illustrates control of a multi-sample compositeliquid cell using electrostatic forces.

FIG. 21 schematically illustrates control of a multi-sample compositeliquid cell using directional air control/

FIG. 22 schematically illustrates static control spur anchoring amulti-sample composite liquid cell.

FIG. 23 is a photograph of a multi-sample composite liquid cell with acentral control volume used to array the original samples of thecomposite liquid cell.

FIGS. 24A-24D schematically illustrate a unit hydrophobic stabilisationfeature for a composite liquid cell.

FIGS. 25A-25D schematically illustrate a number of different hydrophobicstabilisation feature shapes for a composite liquid cell.

FIG. 26 schematically illustrates two hydrophobic stabilisation featuresfor a single composite liquid cell.

FIGS. 27A-27B schematically illustrate positioning of composite liquidcells at discrete locations along a hydrophobic spar with stabilisationfeatures using a control tube and variable flow direction.

FIG. 28 schematically illustrate an array of hydrophobic stabilisationfeatures.

FIG. 29 schematically illustrate a transportation mechanism of acomposite liquid cell using a hydrophobic control surface with astabilisation feature.

FIGS. 30A and 30B schematically illustrate a transportation mechanismfor an array of composite liquid cells using hydrophobic stabilisationfeatures.

FIG. 31 is a diagram illustrating a simple composite liquid cellnetwork.

FIGS. 32A-32F are diagrams illustrating a transportation method for acomposite liquid cell within a composite fluid network.

FIGS. 33A-33E are diagrams illustrating a mixing process of compositeliquid cells within a composite fluid network.

FIGS. 34A and 34B are photographs showing merging of two compositeliquid cells within a composite fluid network.

FIGS. 35A-35C are photographs showing merging of two composite liquidcells to generate a multi-sample composite liquid cell within acomposite fluid network.

FIGS. 36A-36E schematically illustrate a mixing process of compositeliquid cells to generate a multi-sample composite liquid cell within acomposite fluid network.

FIGS. 37A-37C schematically illustrate a composite fluid network forfour composite liquid cells to be combined in two stages.

FIGS. 38A-38G schematically illustrate a composite fluid network for 32composite liquid cells to be combined in five stages.

FIG. 39 schematically illustrates a composite liquid cell apparatus forisothermal nucleic acid amplification.

FIG. 40 schematically illustrates a range of stabilisation features on adisc-shaped hydrophobic platform.

FIG. 40A shows an exemplary system using the disc-shaped platform.

FIGS. 41A-41D are diagrams illustrating generation of a composite fluidnetwork.

FIGS. 42A-42D is a diagram illustrating a hydrophobic control surfacefor immiscible buffer encapsulating fluid path control.

FIGS. 43-47 illustrate various methods that can be implemented ascontroller programming.

DETAILED DESCRIPTION

The invention provides in some embodiments systems and methods for thegeneration of a biological sample within an immiscible fluid cell andpositioned on a free surface of a mutually immiscible carrier fluid.This involves generation, and/or location control, and/or movementcontrol, and/or mixing, and/or processing of biological samples withinsuch composite liquid cells (synonymous with “composite fluid cell”) andpositioned on an immiscible carrier fluid.

The biological sample typically has a density between that of thecarrier fluid and the outer fluid of the composite liquid cell. Thecarrier fluid typically has a density higher than that of the outerfluid of the composite liquid cell.

Typical values of densities for the fluids involved range within thevalues 1,300 to 2,000 kg/m3 for the carrier fluid, 700 to 990 kg/m3 forthe immiscible fluid cell and 900 to 1200 kg/m3 for the biologicalsample. An example of one such set of operating fluids and densities isoutlined herein but is not limited to these; carrier fluid is FluorinertFC-40 (fluorocarbonated oil) density of approximately 1,900 kg/m3; outerfluid of the composite liquid cell is phenylmethylpolysiloxane (siliconeoil) density of approximately 920 kg/m3; and the biological sample is anaqueous based solution of PCR reagents with a density of approximately1000 kg/m3.

In another embodiment the carrier fluid is a perfluorinated amine oil.

In another embodiment, the encapsulating fluid is a solution of aPhenylmethylpolysiloxane-based oil and a polysorbate additive. Theadditives have a hydrophilic-lipophilic balance number in the range of 2to 8. The combined total hydrophilic-lipophilic balance number of theadditives is in the range of 2 to 8. Examples of polysorbate additivesare SPAN 80, SPAN 65 and Tween 20 but are not limited to these. Theseadditives within the buffer encapsulating fluid range between 0.001% and10%.

In another embodiment, the target sample is a solid particle suspensionin aqueous media and the encapsulating fluid is aPhenylmethylpolysiloxane-based oil, on a carrier fluid which is afluorocarbon-based oil.

In another embodiment the target sample is an aqueousmedia-in-Phenylmethylpolysiloxane-based oil and the encapsulating fluidis a Phenylmethylpolysiloxane-based oil, on a carrier fluid which is afluorocarbon-based oil.

In some embodiments the control surfaces are a hydrophobic material.

A system used for making and manipulating composite liquid cells willtypically include a liquid handling system under the control of acontroller (such as a programmable computer). The controller istypically programmed to cause the liquid handling system to carry outvarious steps, with the program steps stored in a nontransitorycomputer-readable medium.

Generating Composite Liquid Cells

Referring to FIG. 1A, a biological sample 1 and a mutually immisciblefluid cell 2 positioned on a free surface of a mutually immisciblecarrier fluid 3 can be combined using a control surface 4. In oneembodiment, the control surface 4 uses electrostatic forces to controlthe location of the immiscible fluid cell 2. The control surface ischarged and is brought in close proximity to the immiscible fluid cell,a charge separation will occur. For example the control surface is givena highly positive charge, negatively charged ions within the immisciblefluid cell will separate towards the charged body. The result is a polarcharge separation and an attractive force towards the charged body.Referring to FIG. 1B a composite liquid cell 5 is generated.

In another embodiment, referring to FIG. 2A a biological sample 21 and amutually immiscible fluid cell 20 positioned on a free surface of amutually immiscible carrier fluid 22 can be combined using a controlsurface 23. The control surface 23 uses the hydrophobic effect tocontrol the location of the immiscible fluid cell 20. Hydrophobicsurfaces repel aqueous based media but silicon-based oils readily wetthe surfaces permitting control using capillary tension. Bringing thecontrol surface 23 into contact with the immiscible fluid cell 20 willresult in a wetting of the surface of the body by the immiscible fluidcell 20. The fluid cell can then be transported to a location on thecarrier fluid 22 by translating the control surface 23. Referring toFIG. 2B a composite cell 24 is generated.

In another embodiment, referring to FIG. 3A a biological sample 31 and amutually immiscible fluid cell 30 positioned on a free surface of amutually immiscible carrier fluid 32 can be combined using a directionalcontrol tube 33. The directional control tube 33 provides an air jetwhich when directed to impinge on the immiscible fluid cell 30 generatesa drag force larger than the translational resistance, therebytransporting the cell fluid in a controlled manner. Referring to FIG. 3Ba composite liquid cell 34 is generated.

In another embodiment, referring to FIG. 4A composite liquid cells canbe generated using the method and system shown. Referring to FIG. 4A awell plate 41 contains biological sample 42 in one or more locations(B1, B2, B3) and is covered by an immiscible fluid 43. A control tube 44has a controllable pressure across the tube. In this mode of operation acontinuous pressure drop is held within the tube, thereby withdrawingthe immiscible fluid 43 into the tube when the tube is translated intocontact with the immiscible fluid 43. A volume of immiscible fluid 43 iswithdrawn into the tube. Referring to FIG. 4B the control tube 44translates into the biological sample 41 and withdraws a volume ofbiological sample C1. Referring to FIG. 4C the control tube returns tothe immiscible fluid layer withdrawing a volume of immiscible fluid.Following this, the control tube then exits the fluid overlay andwithdraws air prior to repeating the procedure at either the samebiological sample location or at a new biological sample location.Referring to FIG. 4D the control tube is loaded with biological samplesC1, C2, C3, immiscible fluid and an air gap 45 separating the immisciblefluids and biological samples. Referring to FIG. 4E the control tube ispositioned over the carrier oil layer 46 housed in a biocompatiblecontainer 47. The control tube may either be in contact with orpositioned between 0-3 mm above the free surface of the carrier oil 46.The flow direction is reversed in the control tube and immiscible fluid,sample and immiscible fluid are deposited on the free surface of thecarrier oil, generating a composite liquid cell. Referring to FIG. 4Fupon depositing a complete composite liquid cell 48 the control tubetranslates to a new position to deposit the next composite liquid cell.

Transporting Composite Liquid Cells

In another embodiment, referring to FIG. 5 the composite liquid cell 51is transported on an immiscible carrier fluid 52 using control surface53. The control surface 53 uses electrostatic forces to control thelocation of the composite liquid cell 52. The control surface is chargedand is brought in close proximity to the outer fluid of the compositeliquid cell, a charge separation will occur. For example the controlsurface is given a highly positive charge, negatively charged ionswithin the immiscible fluid cell will separate towards the charged body.The result is a polar charge separation and an attractive force towardsthe charged body. Using this transport motion one or more compositeliquid cells can be merged or additional biological samples can be addedto a composite liquid cell.

In another embodiment, referring to FIG. 6A a composite liquid cell 61positioned on a free surface of a mutually immiscible carrier fluid 62can be transported using a control surface 63. The control surface 63uses the hydrophobic effect to control the location of the compositeliquid cell 61. Hydrophobic surfaces repel aqueous based media butsilicon-based oils readily wet the surfaces permitting control usingcapillary tension. Bringing the control surface 63 into contact with thecomposite liquid cell 61 will result in a wetting of the surface of thebody by the outer fluid of the composite liquid cell 61. The compositeliquid cell can then be transported to a location on the carrier fluid62 by translating the control surface 63. Using this transport motionone or more composite liquid cells can be merged or additionalbiological samples can be added to a composite liquid cell.

In another embodiment, referring to FIG. 7 a composite liquid cell 71positioned on an immiscible carrier fluid 72 can be position controlledusing a directional control tube 73. The directional control tube 73provides an air jet which when directed to impinge on the compositeliquid cell 71 generates a drag force larger than the translationalresistance, thereby transporting the cell fluid in a controlled manner.Using this transport motion one or more composite liquid cells can bemerged or additional biological samples can be added to a compositeliquid cell.

In another embodiment, referring to FIG. 8A a composite liquid cell 81positioned on an immiscible carrier fluid 82 can be temporarily anchoredusing a hydrophobic spur 83 attached to a base 84. Using this transportmotion one or more composite liquid cells can be merged or additionalbiological samples can be added to a composite liquid cell.

In another embodiment, a composite liquid cell can be moved using acombination of electrostatic forces and the hydrophobic effect.Referring to FIGS. 9A and 9B, a composite liquid cell 91 is positionedon an immiscible carrier fluid 92, and located in contact with ahydrophobic track 93. A dynamic controlling surface 94 uses hydrostaticforces to move the composite liquid cell along the defined hydrophobictrack. The controlling surface 94 can also be used to separate thecomposite liquid cell from the hydrophobic spar and move the compositeliquid cell independently or to another hydrophobic location. Using thistransport motion one or more composite liquid cells can be merged oradditional biological samples can be added to a composite liquid cell.

In another embodiment the hydrophobic spars are partially submerged inthe carrier fluid.

In another embodiment there are multiple controlling surfaces, allowingfor independent motion of discrete composite liquid cells.

In another embodiment, the transport motion is a combination of dynamiccontrol using the hydrophobic effect. A composite liquid cell ispositioned on an immiscible carrier fluid and located in contact with ahydrophobic track. A dynamic controlling surface using the hydrophobiceffect moves the composite liquid cell along the defined hydrophobictrack. The controlling surface can also be used to separate thecomposite liquid cell from the hydrophobic spar and move the compositeliquid cell independently or to another hydrophobic location. Using thistransport motion one or more composite liquid cells can be merged oradditional biological samples can be added to a composite liquid cell.

In another embodiment, the carrier fluid is a continuously flowing fluidand with it's resulting momentum transporting the composite liquid cellsalong its streamlines. In another embodiment the carrier fluid momentumis assisted by static hydrophobic surfaces along which the compositeliquid cells can progress. In another embodiment the carrier fluidmomentum is assisted by dynamic hydrophobic surfaces by which thecomposite liquid cells can be transported.

Unless otherwise stated, any of the disclosure herein related tocomposite liquid cells generally, also applies to multi-sample compositeliquid cells in particular.

Generating Composite Liquid Cells with Multiple Samples

In one embodiment, referring to FIGS. 10A-10F, composite liquid cellscan be generated using the method and system shown. Referring to FIG.10A a well plate 41 contains biological sample 42 in one or morelocations (B101, B102, B103) and is covered by an immiscible fluid 43. Acontrol tube 44 has a controllable pressure across the tube. In thismode of operation a continuous pressure drop is held within the tube,thereby withdrawing the immiscible fluid 43 into the control tube 44when the control tube 44 is translated into contact with the immisciblefluid 43. A volume of immiscible fluid 43 is withdrawn into the tube.Referring to FIG. 10B the control tube 44 translates into the biologicalsample 41 and withdraws a volume of biological sample C101. Referring toFIG. 10C the control tube returns to the immiscible fluid layerwithdrawing a volume of immiscible fluid. Following this, the controltube then repeats the procedure at the same biological sample ortranslates while still within the fluid overlay prior to repeating theprocedure at a new biological sample location. Following the withdrawalof the immiscible fluid and biological samples for a multi-samplecomposite liquid cell the control tube then exits the immiscible oil andwithdraws air prior to repeating the procedure for a new compositeliquid cell. Referring to FIG. 10D the control tube is loaded withbiological samples C101, C102, C103 and immiscible fluid for themulti-sample composite liquid cells. Referring to FIG. 10E the controltube is positioned over the carrier oil layer 46 housed in abiocompatible container 47. The control tube may either be in contactwith or positioned between 0-3 mm above the free surface of the carrieroil 46. The flow direction is reversed in the control tube andimmiscible fluid, sample and immiscible fluid are deposited on the freesurface of the carrier oil, generating a multi-sample composite liquidcell. Referring to FIG. 10F upon depositing a complete multi-samplecomposite liquid cell 48 the control tube translates to a new positionto deposit the next multi-sample composite liquid cell.

Referring to FIGS. 11A-1C, a biological sample 201 at one or morelocations (S1, S2) and a mutually immiscible fluid cell 202 at one ormore locations (O1, O2) positioned on a free surface of a mutuallyimmiscible carrier fluid 203 can be combined using a control surface204. The control surface 204 uses electrostatic forces to control thelocation of the immiscible fluid cell 202. The control surface ischarged and is brought in close proximity to the immiscible fluid cell202, a charge separation will occur. For example the control surface 204is given a highly positive charge, negatively charged ions within theimmiscible fluid cell 202 will separate towards the charged body. Theresult is a polar charge separation and an attractive force towards thecharged body. Referring to FIG. 11B a composite liquid cell 205 at oneor more locations (D1, D2) is generated. The control surface 204 useselectrostatic forces to control the location of the composite liquidcell 205. Referring to FIG. 11C a multi-sample composite liquid cell 206is generated by merging two or more composite liquid cells.

FIGS. 12A and 12B are images showing a multi-sample composite liquidcell resulting from this method. In this example, a composite liquidcell comprising of a 2.5 micro-litre volume of distilled water with 2%green dye and 15 micro-litre of immiscible fluidPhenylmethylpolysiloxane-PD5 oil with 5% polysorbate additive-SPAN 80(v/v) was generated. A second composite liquid cell was generated withthe same reagents however a 2% red dye was used in the distilled waterinstead of the green dye. The colours are discernable as distinct shadesof grey in the figures. The two composite liquid cells were merged usinghydrophobic surfaces (the inverted V-shape visible in these images) andlocated within a stabilisation feature on a hydrophobic spar.

In another embodiment, referring to FIGS. 13A-13C, a biological sample220 within a control tube 223 and a mutually immiscible fluid cell 221positioned on a free surface of a mutually immiscible carrier fluid 222can be combined using the hydrophobic effect of a tubular controlsurface 223. The control tube 223 uses the hydrophobic effect to controlthe location of the immiscible fluid cell 221. Hydrophobic surfacesrepel aqueous-based media but silicon-based oils readily wet thesurfaces, permitting control using capillary tension. Referring to FIG.13B, by bringing the control tube 223 into contact with the immisciblefluid cell 221 will result in a wetting of the surface of the body bythe immiscible fluid cell 221. The biological sample 220 can then bereleased to make contact with the immiscible fluid cell 221 on thecarrier fluid 222. As shown in FIG. 13B a composite liquid cell 224 isgenerated. Referring to FIG. 13C, by repeating the procedure withanother biological sample volume, a multi-sample composite liquid cell225 is generated.

In another embodiment, referring to FIGS. 14A and 14B, a biologicalsample 230 within a mutually immiscible fluid cell 231 positioned on afree surface of a mutually immiscible carrier fluid 232 can besubdivided using an ultrasonic surface 235. Referring to FIG. 14B amulti-sample composite liquid cell 236 is generated.

FIGS. 15A and 15B are images showing such a multi-sample compositeliquid cell like the cell 236 of FIG. 14B. In this example, a 100micro-litre volume of distilled water was vortexed in a 500 micro-litreimmiscible fluid cell composed of Phenylmethylpolysiloxane based oil-PD5with 5% polysorbate additive-SPAN 80 (v/v). FIG. 15B used distilledwater with a 2% green dye for the biological sample. FIGS. 15A and 15Bare a 20 micro-litre representative sample.

In another embodiment, a composite liquid cell with multiple samples ofmultiple distinct sample targets is generated. Referring to FIG. 16,four distinct sample targets are emulsified and combined together stablyas a single multi-sample composite liquid cell with multiple internalsample targets. Four individual composite liquid cells were generatedwith 10 micro-litres of distilled water with green dye 2%, blue dye 2%,yellow dye 5%, and no dye in a 500 micro-litre immiscible fluid cellcomposed of Phenylmethylpolysiloxane based oil-PD5 with 5% polysorbateadditive-SPAN 80 (v/v). Following emulsification the composite liquidcells were merged, see FIG. 16. Evident from FIG. 16 is that thedye-free water samples do not become contaminated by the dyed watersamples, showing that there is no transfer between the samples withinthe multi-sample composite liquid cell. The various colours arediscernable as distinct shades of grey.

In another embodiment, referring to FIGS. 17A and 17B, a mutuallyimmiscible fluid cell 240 and two or more biological samples 241,positioned on a free surface of a mutually immiscible carrier fluid 242are combined using a directional control tube 243. The directionalcontrol tube 243 provides an air jet which when directed to impinge onthe samples 241 generates a drag force larger than the translationalresistance, thereby transporting the buffer fluid 240 and the samples241 in a controlled manner. Referring to FIG. 17B a resultingmulti-sample composite liquid cell 244 is generated.

Transporting the Composite Liquid Cells with Multiple Samples

All general methods of transporting composite liquid cells, for examplethose discussed above, are also applicable to multi-sample compositeliquid cells.

Referring to FIGS. 18A-18C, and as explained in reference to FIGS.6A-6C, a multi-sample composite liquid cell 261 positioned on a freesurface of a mutually immiscible carrier fluid 262 can be transportedusing a device having a hydrophobic control surface 263.

In another embodiment the control surface is partially submerged in thecarrier fluid. Referring to FIGS. 19A and 19B, the multi-samplecomposite liquid cell 290 positioned on a free surface of immisciblecarrier 293 is confined between two hydrophobic spars 291. Thehydrophobic spars 291 have stabilisation features 292 within them. Asshown in FIG. 19A these features are used to control the samples withinthe composite liquid cell, and as shown in FIG. 19B the hydrophobicspars can be moved along the carrier fluid 293 stably transporting themulti-sample composite liquid cells 290.

In another embodiment, referring to FIG. 20, and as explained inreference to FIG. 5, the multi-sample composite liquid cell 251 istransported on an immiscible carrier fluid 252 by a device controlsurface 253 using electrostatic forces.

In another embodiment, referring to FIG. 21, and as explained inreference to FIG. 7, a composite liquid cell 271 positioned on animmiscible carrier fluid 272 can be position controlled using adirectional control tube 273 that provides an air jet.

In another embodiment, referring to FIG. 22, and as explained inreference to FIG. 8, a multi-sample composite liquid cell 281 positionedon an immiscible carrier fluid 282 can be temporarily anchored using ahydrophobic spur 283 attached to a base 284.

Internal Control of Multiple Sample Volumes with a Composite Liquid Cell

In one embodiment the internal sample volumes with a multi-samplecomposite liquid cell can be arrayed for 2D analysis. Referring to FIG.23 a large sample volume can be added to the composite liquid cell andpositioned centrally resulting in the original composite liquid cellsamples being arrayed in the annulus for analysis. A 100 micro-litrevolume of distilled water with 2% green dye was vortexed in a 500micro-litre immiscible fluid cell composed of Phenylmethylpolysiloxanebased oil-PD5 with 5% polysorbate additive-SPAN 80 (v/v). A 20micro-litre representative sample was segmented and a large un-dyedwater sample, in the order of 10 micro-litres was pipetted into thecentre of the composite liquid cell. The resulting structure is shown inFIG. 23.

In one embodiment the internal samples of a multi-sample compositeliquid cell are recombined using a sorbate additive. Examples ofpolysorbate additives are SPAN 80 and Tween 20 but are not limited tothese. These additives within the buffer encapsulating fluid rangebetween 0.001% and 10%.

Mechanical Features for Stabilisation

Referring to FIGS. 24A-24D, a composite liquid cell comprising of atarget sample 311 encapsulated in an immiscible buffer fluid 312positioned on a free surface of a mutually immiscible carrier fluid 313is stably positioned at a hydrophobic surface 314. The hydrophobicsurface 314 is positioned on or in the mutually immiscible carrier fluid313. The hydrophobic surface 314 with localised stabilisation features315 control the position of the composite liquid cell. The stabilisationfeatures allow for generation, and/or location control, and/or movementcontrol, and/or mixing, and/or splitting, and/or processing ofbiological samples within a composite liquid cell and positioned on animmiscible carrier fluid.

Referring to FIG. 24B in one embodiment the composite liquid cellcomprises of approximately 1.7 micro-litre target sample 301encapsulated in approximately 12 micro-litres immiscible buffer 302positioned on a free surface of a mutually immiscible carrier fluid 303is stably positioned at a hydrophobic feature 404 having an invertedV-shaped surface 304. The hydrophobic surface is positioned on or in themutually immiscible carrier fluid 303. The hydrophobic device or feature304 with localised stabilisation features controls the position of thecomposite liquid cell. In one embodiment the stabilisation feature is anotch of 45 degrees with an face depth of 2.5 mm and a thickness of 1.5mm. This is used to control micro-litre size composite liquid cell.Referring to FIG. 24C an underneath plan of the embodiment is shown.Referring to FIG. 24D the previously described embodiment is shown witha housing 305 having a surrounding wall for retaining the carrierliquid.

Referring to FIGS. 25A-25D, a stabilisation feature has a number ofparameters which are adjusted for a given application. Two of theseparameters are the feature shape and the feature thickness. The shape ofthe feature has an impact on the overall size and location control of aninternal sample 321. Referring to FIG. 25A an inverted ‘V’ shapedstabilisation feature 324 controls the internal sample 321 location withthe tangent points A & B as illustrated. Referring to FIG. 25B a curvedshape stabilisation feature 325 has less control over the internalposition of the sample. The variation in shape allows for customisationof the composite liquid cell ratios of an internal sample 321 toencapsulating buffer fluid 322. Typically, a circular shape can achievea greater sample volume to encapsulating fluid ratio and maintaincontamination-free samples. Referring to FIG. 25C the stabilisationfeature thickness C has an impact on the stabilisation of the compositeliquid cell, allowing for customisation of applications. For a largerthickness, typically greater than 50% of the composite liquid celldiameter the stabilisation properties do not improve. For stationarycontrol or processing of a composite liquid cell, a thickness in therange of 5-50% is sufficient for stabilisation of the composite liquidcell on the free carrier surface 323. The stabilisation feature ispositioned on or in the carrier fluid. Referring to FIG. 25D thestabilisation feature can be tapered for composite liquid cellgeneration, and/or location control, and/or movement control, and/ormixing, and/or splitting, and/or processing of biological samples withina composite liquid cell and positioned on an immiscible carrier fluid.

Referring to FIG. 26 a composite liquid cell 331 positioned on animmiscible carrier fluid 332 is positioned between two hydrophobicsurfaces 333 with location features 334. The stabilisation featuresallow for generation, and/or location control, and/or movement control,and/or mixing, and/or splitting, and/or processing of biological sampleswithin a composite liquid cell and positioned on an immiscible carrierfluid.

Referring to FIG. 28 a network of discrete composite liquid cells can begenerated.

Generating Composite Liquid Cell with Mechanical Features

Composite liquid cells may be generated as described earlier, forexample, FIGS. 4A-D. Referring to FIG. 27A the control tube ispositioned over the carrier oil layer 46 housed in a biocompatiblecontainer 47. The control tube may either be in contact with orpositioned between 0-3 mm above the free surface of the carrier oil 46.The flow direction is reversed in the control tube and immiscible fluid,sample and immiscible fluid are deposited on the free surface of thecarrier oil, at a stabilisation feature on a hydrophobic spar 349,generating a composite liquid cell. Referring to FIG. 27B, upondepositing a complete composite liquid cell 48 the control tubetranslates to a new position along the hydrophobic spar 349 to depositthe next composite liquid cell at the stabilisation feature.

In another embodiment two or more hydrophobic surfaces withstabilisation features are used to control the composite liquid celllocation. The use of stabilisation features on a hydrophobic spar allowfor the generation of composite liquid cells at controlled and discretelocations, therefore improving sample tracking and/or automation and/orprocess control.

Transporting Composite Liquid Cells with Mechanical Features

In some embodiments, referring to FIG. 29 a composite liquid cell 361positioned on a free surface of a mutually immiscible carrier fluid 362can be transported using a control surface 363. The control surface 363uses the hydrophobic effect to control the location of the compositeliquid cell 361. Hydrophobic surfaces repel aqueous based media butsilicon-based oils readily wet the surfaces permitting control usingcapillary tension. Bringing the control surface 363 into contact withthe composite liquid cell 361 will result in a wetting of the surface ofthe body by the outer fluid of the composite liquid cell 361. Thecomposite liquid cell can then be transported to a location on thecarrier fluid 362 by translating the control surface 363. Using thistransport motion, one or more composite liquid cells can be merged oradditional biological samples can be added to a composite liquid cell.

In some embodiments referring to FIGS. 30A and 30B, an array ofcomposite liquid cells 371 positioned on a free surface of a mutuallyimmiscible carrier fluid 372 can be transported using the controlsurface 373. The control surface 373 uses the hydrophobic effect tocontrol the location of the composite liquid cells 371. Hydrophobicsurfaces repel aqueous based media but silicon-based oils readily wetthe surfaces permitting control using capillary tension. The controlsurfaces 373 are separated by no more than 0.5 times the sample diameterto ensure sample location confinement. The composite liquid cell array371 is transported to a location on the carrier fluid 372 by translatingthe control surface 373. Using this transport motion with thestabilisation features the composite liquid cells can be processedindividually, ensuring accuracy in the sample throughout discretelocations and/or referencing of the stabilisation feature location. Thisembodiment prevents uncontrolled merging and/or loss of sample.

Composite Liquid Cell Network

Referring to FIG. 31 a composite liquid cell network consists of atleast two stabilisation regions 381 with a connection region 382. Eachstabilisation region allows for the generation, and/or location control,and/or movement control, and/or mixing, and/or splitting, and/orprocessing of biological samples 383 within a composite liquid cell 384and positioned on an immiscible carrier fluid 385.

In some embodiments referring to FIGS. 32A-32F a composite liquid cellnetwork is used to transport a composite liquid cell. Referring to FIG.32A a composite liquid cell 391 on a carrier oil 392 is located with aset of hydrophobic spars with stabilisation features 393. Referring toFIG. 32B a control tube 394 is positioned at/or in the region of thelocation to which the composite liquid cell 391 is to be moved. Thecontrol tube 394 is positioned above the free surface of the carrierfluid 392 and begins infusing immiscible encapsulating buffer fluid 395.Referring to FIG. 32C the encapsulating fluid 395 moves through thenetwork and merges with the composite liquid cell 391. The control tube394 is stopped and the flow direction reversed. Referring to FIG. 32Dthe composite liquid cell moves from the original stabilisation featurelocation to the new prescribed location. Referring to FIG. 32E when thecomposite liquid cell is at the new location the flow in the controltube 394 is stopped and removed. Referring to FIG. 32F the compositeliquid cell has been transported to a new location for processing.

In another embodiment, two or more composite liquid cells can betransported simultaneously using a controlled injection and withdrawalof encapsulating buffer fluid.

In another embodiment, referring to FIGS. 33A-33E, a composite fluidnetwork is used to transport and merge two composite liquid cells.Referring to FIG. 33A a composite liquid cell 401 on a carrier fluid 402is located within a hydrophobic structure 403. Referring to FIG. 33B animmiscible encapsulating buffer fluid 404 is injected onto the carrierfluid 402 at control location 405. Referring to FIG. 33C the immiscibleencapsulating buffer fluid 404 moves through the network and merges withthe composite liquid cells 401. The infusion of immiscible encapsulatingbuffer fluid 404 is stopped. Referring to FIG. 33D the immiscibleencapsulating buffer fluid 404 is withdrawn from control location 405with the internal samples moving from their original locations to a newlocation. Referring to FIG. 33E when the samples are in the new locationthe immiscible encapsulating buffer fluid 404 flow at control location405 is stopped. The samples merge resulting in a single sample compositeliquid cell. The formation of complex encapsulating oil interfaces,bounded by control surfaces, carrier oil and an air interface, isgoverned by free surface energy which is proportional to the surfacearea of the encapsulating oil/air interface. Controlled withdrawal ofencapsulating oil will cause the system to minimize surface arearesulting in encapsulating oil being removed from the networkextremities equally. This interfacial contraction from the network'sextremities also transports aqueous droplets contained within.

FIG. 34A is a picture of a composite fluid network with two compositeliquid cells. The composite liquid cells in this picture contain 2.5micro-litres sample volumes of distilled water, one sample dyed red andthe second dyed blue. The encapsulate buffer fluid was an immisciblefluid Phenylmethylpolysiloxane-PD5 oil on an immiscible fluorocarbonatedcarrier—FC40. The hydrophobic spar was a PTFE based material which waslocated on the interface of the carrier fluid and air. The stabilisationfeatures have a dimension of approximately 2 mm at the widest and anangle of approximately 45 degrees.

FIG. 34B shows a single composite liquid cell with a single internalsample, which is a result of the merging of the previous two compositeliquid cell sample volumes by the method described previously.

Referring to FIGS. 35A-35C and 36A-36E, the encapsulating fluid has anadditive and the merging of composite liquid cells results in amulti-sample composite liquid cell.

Examples of composite liquid cell networks are outlined herein but arenot limited to these; Referring to FIGS. 37A-37C, a composite liquidcell network for merging four composite liquid cells in two stages.Referring to FIG. 37B the composite liquid cells in the adjoiningstabilisation features merge first. Referring to FIG. 37C the remainingcomposite liquid cell samples are merged resulting in a single compositeliquid cell.

Another example, referring to FIGS. 38A-38G, is a network for merging 32composite liquid cells in 5 stages. Referring to FIG. 38A a network ofhydrophobic spars and carrier oil prior to composite liquid cellloading. Referring to FIG. 38B composite liquid cells are loaded in theouter locations of the composite fluid network. Referring to FIG. 38Cthe composite liquid cells in the adjoining stabilisation features aremerged using an infusion/withdrawing process of the immiscible bufferencapsulating fluid. Referring to FIG. 38D the second stage of thecomposite liquid cell processing—the composite liquid cells in theadjoining stabilisation features are merged using aninfusion/withdrawing process of the immiscible buffer encapsulatingfluid. The composite liquid cells now contain four of the originallydeposited composite liquid cells. Referring to FIG. 38E the third stageof the composite liquid cell processing—the composite liquid cells inthe adjoining stabilisation features are merged using aninfusion/withdrawing process of the immiscible buffer encapsulatingfluid. The composite liquid cells now contain eight of the originallydeposited composite liquid cells. Referring to FIG. 38F the fourth stageof the composite liquid cell processing—the composite liquid cells inthe adjoining stabilisation features are merged using aninfusion/withdrawing process of the immiscible buffer encapsulatingfluid. The composite liquid cells now contain sixteen of the originallydeposited composite liquid cells. Referring to FIG. 38G the fifth stageof the composite liquid cell processing—the composite liquid cells inthe adjoining stabilisation features are merged using aninfusion/withdrawing process of the immiscible buffer encapsulatingfluid. The composite liquid cells now contain all thirty two of theoriginally deposited composite liquid cells. At each of the stagesbiological processing of the composite liquid cells can be performed.These processes may include but are not limited to; polymerase chainreaction, and/or thermal cycling, and/or isothermal amplification,and/or optical analysis, and/or the addition of further reagents.

In another embodiment, referring to FIGS. 41A-41D, a composite fluidnetwork is generated using a method of infusing the immiscibleencapsulating buffer fluid. Referring to FIG. 41A the target samples aredispensed within the network of hydrophobic spars on the carrier fluid.Referring to FIG. 41B the immiscible encapsulating buffer fluid isinfused on the carrier fluid. Referring to FIG. 41C the immiscibleencapsulating buffer fluid encapsulated the sample volumes within thestabilisation features of the hydrophobic spars. Referring to FIG. 41Done embodiment of the composite fluid network is shown.

In another embodiment, referring to FIGS. 42A-42D, the composite fluidnetwork has hydrophobic control surfaces to control the immiscibleencapsulating buffer fluid network path on the carrier fluid. Referringto FIG. 42A a composite fluid network with two samples at discretestabilisation features within an immiscible encapsulating buffer fluid.Referring to FIG. 42B hydrophobic control surfaces are used to shear theimmiscible encapsulating buffer fluid, while maintaining a carrier fluidpath. Referring to FIG. 42C the immiscible encapsulating buffer fluidcan be withdrawn. Referring to FIG. 42D two discrete composite liquidcells are generated. This method is also used to progress a compositeliquid cell through a composite fluid network and/or to isolate acomposite liquid cell from other processes within the composite fluidnetwork.

Processing the Composite Liquid Cells

In certain embodiments, the system may involve one or more of thefollowing steps in any order which achieves the target samplecombination at the end of the method: extracting samples for target;processing the samples for loading into the dispensing system;dispensing the target sample; dispensing the immiscible encapsulatingfluid cell; dispensing the carrier fluid; combining the biologicalsample and immiscible encapsulating fluid cell; combining the biologicalsample and composite liquid cell; combining the biological sample andmulti-sample composite liquid cell; controlling the motion of theimmiscible encapsulating fluid cell; controlling the motion of thecarrier fluid; transporting one or more immiscible fluid cells;transporting one or more immiscible fluid cells to combine with one ormore biological samples; transporting one or more composite liquid cellsto combine with one or more biological samples; transporting one or moremulti-sample composite liquid cells to combine with one or morebiological samples; transporting one or more composite liquid cells tocombine with one or more composite liquid cells; transporting one ormore multi-sample composite liquid cells to combine with one or morecomposite liquid cells; transporting one or more multi-sample compositeliquid cells to combine with one or more multi-sample composite liquidcells; detecting an effect of the biological sample within the compositeliquid cell; detecting an effect of the biological sample within themulti-sample composite liquid cell; detecting effects of biologicalsamples within the multi-sample composite liquid cell; providing outputinformation to a user of the detection; analysing the output data of thedetection. Examples of the biological protocols are given in the ensuingsections.

PCR

Polymerase Chain Reaction (PCR) has been used extensively to amplifytargeted DNA and cDNA for many applications in molecular biology. ThePCR technique amplifies a single or a few copies of a piece of DNA,generating thousands to billions of copies of a particular DNA sequence.Modern PCR instruments carry out the PCR process in reaction volumesranging from 10-200 micro-litres. One of the largest obstacles tocarrying out PCR in small volumes is the difficulty in manipulatingsmall volumes of the constituent reagents with manual pipettes. Anotherobstacle for PCR is the difficulties in multiplexing reactions allowingfor increased thought put.

The methods of the invention generally involve combining the necessaryfluids to form the resulting multi-sample composite liquid cell. In oneembodiment the target sample is an aqueous biological sample, comprisingreagents required for nucleic acid amplification, and the outer fluidcell is a silicone oil (Phenylmethylpolysiloxane, PD 5) with apolysorbate additive (SPAN 80), on a carrier fluid which is afluorocarbon-based oil (Fluorinert FC-40). The individual reagents arearrayed such that all the necessary components for PCR are placed asindividual composite liquid cells. This prevents cross-contamination ofbiological reagents. The individual composite liquid cell components arecombined together in the correct sequence. The individual compositeliquid cells are then combined together forming a multi-sample compositeliquid cell. The multi-sample composite liquid cell is then transportedinto different thermal zones or optical interrogation zones wherequantitative measurement of the products are performed via fluorescentmeasurement. The placement of PCR target volumes within composite liquidcells prevents evaporation during thermal cycling. Typical thermalcycling temperatures range between 55-95° C.

In a further embodiment the composite liquid cells may have anassociated detection mark added as a discrete sample.

In a further embodiment, the combination of post-PCR reactions may berequired for further processing that may include genetic sequencing. Theuse of multi-sample composite liquid cells greatly simplifies thecollection and sequencing procedure for these relatively small targetvolumes. The multi-sample composite liquid cells following individualprocessing can be combined selectively, removing any unspecificamplification reactions or inefficient reaction from the final collectedvolume. The amalgamated final target volume, consisting of manydiffering target molecules, is transferred into a sequencing instrumentfor detailed analysis. The composite liquid cell facilitates 100% volumeretrieval as the biological sample is processing and does not need totouch any solid surface and also has the additional benefit of ananti-wetting characteristic. Additionally, the fluid volume requiringthermal cycling has been greatly reduced—removed the entire mass andthermal resistance of the static well plates—targeted heating strategiescan facilitated lower power instruments and faster reaction processingtimes.

TABLE 1 Fluorescence intensity results showing the ability to detectSingle Nucleotide Polymorphisms in composite liquid cell with nocarryover contamination FAM READINGS RED READINGS < > NTB 1 Allele 1 NTB2 Allele 2 NTB 3 < > NTB 1 Allele 1 NTB 2 Allele 2 NTB 3 A 3776 345033492 4579 2916 A 3489 3460 3291 14928 2685

Referring to Table 1 the composite liquid cell has successfullyperformed Single Nucleotide Polymorphism detection. Two positive sampleswere used, each containing a different allele of the gene, named allele1 and allele 2. Each allele was labelled with a different dye andreading at the correct intensity allowed detection of each allele. A notemplate control was added in a composite liquid cell and amplified.This was repeated in the following order for a positive allele 1, a notemplate control, a positive allele 2 and finally another no templatecontrol. Referring to Table 1 the results show that amplification wassuccessfully performed in composite liquid cells with no crosscontamination between samples. This is indicated by no rise influorescence intensity between the initial no template control and theno template control samples that followed each positive samples.

Referring to FIG. 39 an apparatus for an isothermal nucleic acidamplification of multiple composite liquid cells. The composite liquidcells are arrayed on a circular platform which moves through therequired stages of composite liquid cell generation, thermal processing,optical detection and removal. The composite liquid cell generation hasthree stages, the addition of the immiscible encapsulating buffer fluidto the carrier fluid, the addition of the master PCR reagents to theimmiscible encapsulating buffer fluid and the addition of the sample tothe composite liquid cell. The composite liquid cell is thenbiologically processed by heating to 65° C. for 10 minutes. To completethe rotation of the hydrophobic plate, the composite liquid cells passthough a fluorescent detection region after which the composite liquidcells are removed from the carrier fluid and the platform returns to theinitial composite liquid cell generation stage. The plate continuouslyrotates with new composite liquid cells generated at a speed dependanton the rotation speed of the platform.

FIG. 40 shows an example of a disc-shaped platform, typically of ahydrophobic material, that provides a network of stabilisation features.FIG. 40A shows the disc incorporated in a system. The system has astationary circular bath of carrier liquid. Rotating on the surface ofthis carrier oil bath is a PTFE disk containing stabilization features.The disc may be rotated by a shaft driven by the motor-gearbox assemblyshown. Composite liquid cells may be dispensed into the stabilisationfeatures from a fixed dispensing tube (not shown).

Digital PCR

The Polymerase Chain Reaction (PCR) is a widely used molecularamplification technique. The technique has widespread applications inclinical diagnostics, agricultural biotechnology and bioresearch. It isroutinely used for the detection of SNP's, diagnosis of hereditarydiseases, genetic fingerprinting, forensic analysis, gene expression andother types of nucleic acid analysis. The development of Digital PCR hasincreased the use of conventional PCR. Digital PCR is a technique thatamplifies a single DNA template. For a review of the PCR methodology seeDigital PCR (Proc Natl Acad Sci USA 96(16):9236-41 (1999)) by Vogelsteinand Kinzler, Principle and application of digital PCR (Expert ReviewMolecular Diagnostics 4(1):41-47 (2004)) by Pohl and Shih.

The methods of the invention generally involve combining anddistributing the necessary fluids to form the resulting multi-samplecomposite liquid cell. In one embodiment the target sample is an aqueousbiological sample, comprising reagents required for nucleic acidamplification, and the outer fluid cell is a silicone oil(Phenylmethylpolysiloxane, PD 5) with a polysorbate additive (SPAN 80),on a carrier fluid which is a fluorocarbon-based oil (Fluorinert FC-40).The individual reagents are arrayed such that all the necessarycomponents for digital PCR are placed as individual composite liquidcells. This prevents cross-contamination of biological reagents. Theindividual composite liquid cell components are combined together in thecorrect sequence. The individual composite liquid cells are thenultrasonicated to form a multi-sample composite liquid cell. Themulti-sample composite liquid cell is then transported into differentthermal zones or optical interrogation zones where quantitativemeasurement of the products are performed via fluorescent measurement.The placement of digital PCR target volumes within composite liquidcells prevents evaporation during thermal cycling. Typical thermalcycling temperatures range between 55-95° C. The multi-sample compositeliquid cell facilitates the use of simpler detection methods as themulti-sample composite liquid cell does not require optics for threedimensional integration for quantification determination.

In another embodiment the individual composite liquid cells aremechanically agitated to form a multi-sample composite liquid cell.

Nucleic Acid/DNA Ligation

Nucleic acid ligation involves the use of nucleic acid ligases, whichare enzymes that are used to join fragments of nucleic acid together. Inconstructing long DNA strands multiple shorter DNA fragments arecombined together, it is therefore necessary to perform multipleligation steps to achieve these results. The product of one ligationreaction becomes the fragment of another. The ligation must be performedin a pairwise fashion to avoid efficiency and orientation problemsaffecting the reaction.

In one embodiment, the reaction reagents—the DNA fragments to be joined;the ligase enzyme; and the buffer reagents—are pipetted individuallyinto separate wells of a microtitre plate. The outer composite fluid, asilicone oil (Phenylmethylpolysiloxane, PD 5), is pipetted on top of theaqueous reagents within each well, creating an oil-aqueous interface.

A hydrophobic sampling capillary manipulated by an automated roboticplatform, aspirates a volume of oil, in the range of 500-2000 nanolitresof silicone oil (Phenylmethylpolysiloxane, PD 5) from the first samplingwell, followed by aspirating 100-700 nl of the target volume (aqueousreagents), followed by a similar volume of silicone oil(Phenylmethylpolysiloxane, PD 5) in the range of 500-2000 nanolitres.The sampling capillary then translates through the air, stillaspirating, into the next sampling well. The procedure is repeated forthe next sampling well, aspirating the covering oil, followed by theaqueous reagents, followed by the covering oil. The hydrophobic samplingcapillary tube then translates until a predetermined number of sampleshave been aspirated. The hydrophobic sampling capillary now has a seriesof discrete liquids segmented by air, aspirated during translation.

The hydrophobic sampling capillary tube is translated over theprocessing platform. The direction of flow in the hydrophobic samplingcapillary tube is reversed to dispense each fluid sequence as anindividual composite liquid cell on to the surface of a carrier oil, afluorocarbon-based oil (Fluorinert FC-40). The hydrophobic samplingcapillary tube is translated between discreet liquid dispensing to a newcomposite liquid cell initial location. The composite liquid cells aredispensed such that they interface with a hydrophobic spar which securesthe composite liquid cells location. The sequence of composite liquidcells combining to give the final DNA synthetic construction is placedon a single hydrophobic spar. The dispensing process generates a seriesof composite liquid cells separated by a distance in the range of 0.5mm-10 mm, positioned on hydrophobic spars.

In the case of single-sample composite liquid cells, a control surfacemanipulated by an automated robotic platform creates a pairwisecombinations of composite liquid cells such that the reagents withincomposite liquid cells combine and mix via the action of capillarytension with the control surface. Or, if a fluid cell network is beingused, then the injection/withdraw immiscible encapsulating buffer fluidcontrols of the composite fluid network make pairwise combinations ofcomposite liquid cells such that the reagents within composite liquidcells combine and mix.

These reagents contain the neighbouring DNA fragments and other reagentsnecessary for ligation to occur. The composite liquid cells arecontrolled at specific temperature conditions for a specific time forligation to occur. Typical conditions are 16° C. for 1 hour to ensureligation of cohesive-ended fragments. After this ligation step, furtherpairwise combinations of neighbouring composite liquid cells are formedand processed at the correct temperature generating longer fragments.This process is repeated until the desired final fragment length isreached. The newly constructed synthetic DNA strand is then aspiratedand stored for future use.

The creation of composite liquid cells greatly simplifies the method ofDNA ligation. The smaller reaction volume, not normally used in thisprocess ensures higher reaction efficiency and faster reaction times.The combination of target DNA strand fragments for each ensuing pairwiseligation is greatly simplified and the number of overall liquidmanipulations is greatly reduced, as the entire sequence of target DNAcomposite liquid cells are co-located on the hydrophobic spar network.Coalescence by this method is easy to achieve by manipulating theposition of the composite liquid cells. This method of ligation isparticularly useful where the products of one ligation step arenecessary for another step.

The use of composite liquid cells greatly simplifies the collectionprocedure for these relatively small target volumes. The compositeliquid cells following individual processing can be combinedselectively, removing any non-reactive sample or inefficient reactionsfrom the ligation process. The composite liquid cell facilitates 100%volume retrieval as the biological sample in processing does not need totouch any solid surface and also has the additional benefit of ananti-wetting characteristic.

More particularly, multi-sample composite liquid cells may be used. Themulti-sample composite liquid cells are controlled at specifictemperature conditions for a specific time for ligation to occur.Typical conditions are 16° C. for 1 hour to ensure ligation ofcohesive-ended fragments. Following ligation nucleic acid amplificationis performed to selectively amplify the correct region of interest andnext ligation step. After this step, internal samples are combined in apairwise sequence, additional reagents are added if required andprocessed at the correct temperature generating longer fragments. Thisprocess is repeated until the desired final fragment length is reached.The newly constructed synthetic DNA strand is then aspirated and storedfor future use.

The creation of multi-sample composite liquid cells greatly simplifiesthe method of DNA ligation. The smaller reaction volume, not normallyused in this process ensures higher reaction efficiency and fasterreaction times. The combination of target DNA strand fragments for eachensuing pairwise ligation is greatly simplified and the number ofoverall liquid manipulations is greatly reduced, as the entire sequenceof target DNA samples are co-located within the one multi-samplecomposite liquid cell. Internal sample coalescence by this method iseasy to achieve by manipulating the outer oil surface positions withinthe location features of the hydrophobic surface. This method ofligation is particularly useful where the products of one ligation stepare necessary for another step.

Genetic Sequencing Bead Coating

Genetic sequencing bead preparation is a process by which small beadsare coated in an application-specific chemistry. In one embodiment thecoating of beads in advance of genetic screening is achieved bygenerating composite liquid cells with an aqueous solution of beads asthe target volumes. The specific primer chemistry, used to coat thebeads, is introduced into the fluid cell via a capillary depositing anaqueous droplet directly into the fluid cell, such that the targetvolumes combine, resulting in mixing and coalescence of the primerchemistry with the aqueous bead solution.

In another embodiment, there is creation of a composite liquid cell forthe primer chemistry and then manipulation to coalesce with thecomposite liquid cell containing the aqueous bead solution, such thatthe target volumes combine and mix.

These methods provide for a convenient way of manipulating and combiningsub-microlitre volumes of fluid that is currently not possible toachieve using conventional techniques, thereby reducing the initialsample volumes and improving the bead coating efficiency by reducing thereaction volume. Further processing using PCR and thermal cycling andgenetic sequencing is application-specific.

The use of composite liquid cells greatly simplifies the collectionprocedure for these relatively small target volumes. The compositeliquid cells following individual processing can be combinedselectively, removing any non-reactivated beads. The composite liquidcell facilitates 100% volume retrieval as the biological sample inprocessing does not need to touch any solid surface and also has theadditional benefit of an anti-wetting characteristic. These featuresmake automation of the biochemistry process easier to facilitate.

The invention is not limited to the embodiments described but may bevaried in construction and detail. For example the encapsulating bufferfluid may be any suitable liquid or gas, most commonly a liquid. Thecarrier fluid may be any suitable liquid or gas, but most commonly aliquid. The choice of encapsulating and carrier fluid may be chosen suchthat the functional groups of the fluids result in mutual immiscibilitythat limits diffusion of carrier fluid molecules into the encapsulatingfluid and vice versa. This immiscibility constraint must also applybetween the encapsulating fluid and the target sample such thatmolecular diffusion is limited by the differing functional groups of theconstituent fluids. For example the phenylmethyl functional groupspresent in phenylmethylpolysiloxanes (silicone oils) chosen forencapsulating fluids are immiscible with the perfluoro functional groupspresent in fluorocarbon based carrier oils that are commonly chosen ascarrier fluids. These two oils are furthermore immiscible with aqueousbased samples that are common in molecular biochemistry. It is alsoadvantageous to choose fluids of differing densities such that thecarrier fluid has the highest density and forms the lowest layer offluid, the encapsulating fluid has the lowest density and the targetsample has an intermediate density between that of the carrier fluid andthe encapsulating fluid.

Embodiments

Some embodiments of the invention encompasses a sample handling systemcomprising a sample-liquid input, an encapsulating-liquid input, acarrier-liquid conduit comprising a stabilisation feature, aliquid-handling system, and a controller operably connected to theliquid-handling system. In some embodiments the controller may beprogrammed to: (1) draw an encapsulating liquid from theencapsulating-liquid input; (2) discharge the drawn encapsulating liquid(a) onto a free surface of a carrier liquid in the carrier-liquidconduit and (b) proximate to the stabilisation feature, theencapsulating liquid being immiscible with the carrier liquid, so thatthe discharged encapsulating liquid does not mix with the carrierliquid, floats on top of the carrier liquid, and is immobilised by thestabilisation feature; (3) draw a sample liquid from the sample-liquidinput; and (4) discharge the drawn sample liquid, the sample liquidbeing immiscible with the encapsulating liquid and with the carrierliquid, so that the sample liquid does not mix with the encapsulatingliquid or with the carrier liquid. Exemplary flowcharts are shown inFIGS. 43-47.

In some embodiments the liquid handling system comprises a control tubeand a driver. In some embodiments the controller may be programmed toactuate the driver to cause the control tube to carry out steps (1) and(3) before carrying out steps (2) and (4) (FIG. 44). In some embodimentsthe controller may be programmed to actuate the driver to cause thecontrol tube to carry out step (1), then step (3), then to (5) drawadditional encapsulating liquid, then to (6) discharge the encapsulatingliquid, then to carry out step (4), then step (2), so that theencapsulating liquid, the sample liquid, and the additionalencapsulating liquid are discharged as a unit from the control tube andonto the free surface of the carrier liquid in the carrier-liquidconduit, the encapsulating liquid and the additional encapsulatingliquid thereby merging and surrounding the sample liquid to form acomposite liquid cell (FIG. 45). In some embodiments the controller mayalso be programmed to actuate the driver to cause the control tube,after step (5) and before step (6), to (7) draw a separator (FIG. 45).In some embodiments the separator may comprise air. In some embodimentsthe controller may be programmed to actuate the driver to cause thecontrol tube, after step (7) and before step (6), to (1 a) draw anencapsulating liquid from an encapsulating-liquid input, then (3 a) drawa sample liquid from a sample-liquid input, then (5 a) draw additionalencapsulating liquid, then (6 a) discharge the encapsulating liquid ofsteps (1 a) and (5 a) with the sample liquid of step (3 a) as a secondunit from the control tube and onto the free surface of the carrierliquid in the carrier-liquid conduit, the second unit thereby forming asecond composite liquid cell (FIG. 45).

In some embodiments the controller is further programmed to actuate thedriver to cause the control tube, after step (5) and before step (6), to(8) draw a second sample liquid from a second sample-liquid input, thesecond sample liquid being immiscible with the carrier liquid and theencapsulating liquid, then (9) draw additional encapsulating liquid,then (10) discharge the additional encapsulating liquid of step (9) andthe second sample liquid into the unit, so that the composite liquidcell thereby formed comprises a droplet of the sample liquid and adroplet of the second sample liquid (FIG. 46).

In some embodiments the liquid-handling system comprises a control tubeand a driver, and the controller is programmed to actuate the driver tocause the control tube to carry out steps (1) through (4) in the orderrecited (FIG. 47). In some embodiments the controller may be programmedto perform step (1), then repeat step (2) for a plurality ofstabilization features, then perform step (3), then repeat step (4) forthe plurality of stabilization features, thereby forming a plurality ofcomposite liquid cells distributed among the stabilization features(FIG. 47). In some embodiments the controller is further programmed toactuate the driver to cause the control tube to (11) draw a reagent thatis miscible with the sample liquid, and (12) discharge the reagentproximate to the discharged sample liquid (FIG. 47). In some embodimentsthe sample handling system further comprises a motion system thattranslates at least a discharging portion of the liquid-handling systemrelative to the carrier-liquid conduit. In some embodiments thecontroller may be programmed to actuate the motion system to cause thecontrol tube to move a composite liquid cell formed on the free surfaceof the carrier liquid relative to the carrier liquid conduit.

In some embodiments, (a) the carrier-liquid conduit comprises a bathsized to receive a disc rotatable therewithin upon a bath of carrierliquid; (b) the stabilisation feature is formed in the disc; (c) thesystem further comprises (1) a rotation driver operably coupled to thedisc to cause it to rotate in the bath, and (2) a motion system thattranslates at least a discharging portion of the liquid-handling systemvertically relative to the carrier-liquid conduit; and (d) thecontroller is operably connected to the rotation driver and to themotion system and is programmed to cause the rotation system to rotatethe disc and to cause the motion system to translate the dischargingportion of the liquid-handling system vertically relative to the disc.

In some embodiments the controller may be programmed to cause theliquid-handling system to discharge sufficient encapsulating liquidbetween two composite liquid cells, formed on the free surface of thecarrier liquid and separated by a gap, liquid to bridge the gap, therebycausing the two composite liquid cells to merge with one another. Insome embodiments the controller may be programmed to cause theliquid-handling system to discharge the sample liquid of step (4)proximate to the discharged encapsulating liquid.

In some embodiments the invention encompasses a sample handling methodcomprising drawing an encapsulating liquid from an encapsulating-liquidinput; discharging the drawn encapsulating liquid (a) onto a freesurface of a carrier liquid in a carrier-liquid conduit comprising astabilisation feature and (b) proximate to the stabilisation feature,the encapsulating liquid being immiscible with the carrier liquid, sothat the discharged encapsulating liquid does not mix with the carrierliquid, floats on top of the carrier liquid, and is immobilised by thestabilisation feature; drawing a sample liquid from a sample-liquidinput; and discharging the drawn sample liquid, the sample liquid beingimmiscible with the encapsulating liquid and with the carrier liquid, sothat the sample liquid does not mix with the encapsulating liquid orwith the carrier liquid.

In some embodiments the invention encompasses a method for processingbiological samples, the method comprising encapsulating a sample in animmiscible buffer fluid and moving them as a combined unit for samplehandling.

In some embodiments, the sample, while encapsulated, is placed on or ina carrier fluid.

In some embodiments, the carrier fluid is a liquid and the sample isplaced on the surface of the liquid, the carrier fluid being immisciblewith the encapsulating buffer fluid.

In some embodiments, the carrier fluid has a higher density than theencapsulating buffer fluid.

In some embodiments, the encapsulating fluid is non-reactive with thetarget sample.

In another embodiment, there is a control surface which controls themotion of the encapsulating buffer fluid through electrostatic forcessuch as for introducing samples into the buffer fluid.

In some embodiments, there is a control surface which controls themotion of the encapsulating buffer fluid through surface tension forcessuch as for introducing samples into the buffer fluid.

In some embodiments, there are a plurality of controlling surfaces.

In some embodiments, the controlling surfaces include a dynamic surface.

In some embodiments, the controlling surfaces include a static surface.

In some embodiments, the controlling surfaces include a combination ofdynamic and static surfaces.

In some embodiments, the controlling surfaces are submerged within thecarrier fluid at some times.

In some embodiments, the controlling surfaces are on the carrier fluidinterface.

In some embodiments, the controlling surfaces are above the carrierfluid.

In some embodiments, the carrier fluid flows.

In some embodiments, at least one analysis system performs analysis ofthe biological sample within the encapsulating buffer fluid on thecarrier fluid.

In some embodiments, there is a plurality of analysis stages.

In some embodiments, the analysis includes thermal cycling theencapsulating buffer fluid.

In some embodiments, the buffer encapsulating fluid is a silicone-basedoil or a fluorocarbon-based oil.

In some embodiments, the carrier fluid is a silicone-based oil or afluorocarbon-based oil.

In some embodiments, the sample is a biological sample.

In some embodiments, the sample is an aqueous-based biological sample.

In some embodiments, the sample is a solid particle.

In some embodiments, the sample is an aqueous-in-oil emulsion.

In some embodiments, sample amplification is performed, and in somecases quantification of the amplification is performed.

In some embodiments, real time quantification of the amplification isperformed.

In some embodiments, the method comprises forming a target sampleencapsulated in an immiscible fluid volume and depositing it on acarrier fluid which is immiscible with the encapsulating buffer fluid;and controlling the encapsulated buffer fluid with electrostatic forces.

In some embodiments, the target sample is encapsulated in an immisciblefluid volume dispensed into a flowing carrier fluid.

In some embodiments, the encapsulated sample is dispensed into a staticlocation.

In some embodiments, biological processing of the encapsulated sample isperformed, and the encapsulated sample is combined with one or moreencapsulated samples.

In some embodiments, genomic amplification is performed.

In some embodiments, the method comprises the step of controlling theencapsulating buffer fluid with surface tension forces.

In some embodiments, nucleic acid ligation is performed.

In another aspect, the invention provides a sample handling systemcomprising means for performing the steps of any method as definedherein.

In some embodiments, the system comprises: a conduit for continuous flowof the carrier fluid such as oil which carries the encapsulating fluidwith the target sample; an analysis stage; and a controller to controlthe system.

In some embodiments, the system comprises a thermal cycling stage.

In some embodiments, the system is adapted to deposit the encapsulatedinto or onto a static position on a carrier fluid.

In some embodiments, herein there are a plurality of positions.

In some embodiments, there are a plurality of conduits.

In some embodiments, the system further comprises means for controllingmovement of an encapsulated sample by electrostatic forces.

In some embodiments, the system is adapted to move an encapsulatedsample onto or on a static carrier fluid.

In some embodiments, the sample is moved to any of a plurality oflocations and there may be a plurality of samples on the static carrierfluid.

In some embodiments, a conduit is closed.

In some embodiments, the invention encompasses a method for processingsamples, in some cases biological samples, the method comprisingencapsulating two or more samples in an buffer fluid immiscibly with thesamples and moving them as a combined unit.

In some embodiments the buffer fluid may be a liquid.

In some embodiments, two or more samples, while encapsulated, are placedon or in a carrier fluid.

In some embodiments, the sample is a multiplex reaction.

In some embodiments, two or more samples are encapsulated within the oneencapsulant fluid surface.

In some embodiments, the carrier fluid is a liquid and the sample orsamples, while encapsulated are placed on the surface of the liquid, thecarrier fluid being immiscible with the encapsulating buffer fluid.

In some embodiments, the sample, while encapsulated, is combined withanother sample, while encapsulated, resulting in two discrete samples,while encapsulated within one encapsulating surface.

In some embodiments, the sample, while encapsulated, will be processedfor one or more targets.

In some embodiments, at least one analysis system performs analysis ofone or more biological samples within the encapsulating buffer fluid, insome cases on the carrier fluid.

In some embodiments, the buffer encapsulating fluid has a additive addedfor sample stability.

In some embodiments, the buffer encapsulating fluid is a silicone-basedoil with a polysorbate additive.

In some embodiments, the polysorbate additive is added in the range of0.001% to 10%.

In some embodiments the buffer fluid includes an additive including asurfactant.

In some embodiments, the total hydrophilic-lipophilic balance number ofthe added surfactants are in a range of 2 to 8.

In some embodiments, the method comprises forming two or more targetsamples encapsulated in an immiscible fluid volume and depositing it ona carrier fluid which is immiscible with the encapsulating buffer fluid;and controlling the encapsulated buffer fluid with electrostatic forces.

In some embodiments, two or more target samples are encapsulated in animmiscible fluid volume dispensed into a flowing carrier fluid.

In some embodiments, at least one encapsulated sample is dispensed intoa static location.

In some embodiments, biological processing of at least one encapsulatedsample is performed, and at least encapsulated sample is combined withone or more other encapsulated samples.

In some embodiments a further sample may be added to the cell therebyarraying samples.

In some embodiments the samples are arrayed for optical detection.

In some embodiments the cell is transported by impingement of a gas froma direction al outlet such as a tube.

In some embodiments, the invention provides a sample handling systemcomprising means for performing the steps of any method as definedabove.

In some embodiments, the system comprises: a conduit for continuous flowof the carrier fluid such as oil which carries the encapsulating fluidwith two or more target samples; an analysis stage; and a controller tocontrol the system.

In some embodiments the system comprises: a conduit for flow of a bufferfluid such as oil with two or more target samples which is interfacedwith a carrier fluid; an analysis stage; and a controller to control thesystem.

In some embodiments the system comprises: a moving hydrophobic spar onwhich a carrier fluid such as oil carries buffer fluid with two or moretarget samples; an analysis stage; and a controller to control thesystem.

In some embodiments, the carrier oil has a surfactant additive.

In some embodiments the invention is used for processing a sample, thesample being encapsulated in an immiscible buffer fluid and positionedat a hydrophobic control surface for sample handling.

In some embodiments, the hydrophobic control surface is stationary.

In some embodiments, the hydrophobic control surface is dynamic.

In some embodiments, the hydrophobic control surface is a fluoropolymer.

In some embodiments, there are a plurality of hydrophobic controllingsurfaces.

In some embodiments, the controlling surface is electrostaticallyenergised.

In some embodiments, the hydrophobic controlling surfaces include acombination of dynamic and static surfaces.

In some embodiments, the composite liquid cell is controlled by one ormore hydrophobic controlling surfaces.

In some embodiments, the hydrophobic control surface is temperaturecontrolled.

In some embodiments, the hydrophobic control surface is part of anoptical detection system.

In some embodiments, the hydrophobic control surface has a marker foroptical detection.

In some embodiments, the hydrophobic control surface has a radiofrequency identification circuit.

In some embodiments, the hydrophobic control surface controls aplurality of composite liquid cells.

In some embodiments, the hydrophobic control surface has stabilisationfeatures.

In some embodiments, there are a plurality of stabilisation features.

In some embodiments, the stabilisation features are pockets into thehydrophobic control surface.

In some embodiments, the stabilisation features are v shaped.

In some embodiments, the stabilisation features are circular shaped.

In some embodiments, the stabilisation features are tapered.

In some embodiments, the stabilisation features are used to locate thesample encapsulated in an immiscible buffer fluid on a mutuallyimmiscible carrier fluid.

In some embodiments, the stabilisation features are arrayed to form anetwork.

In some embodiments, the network is used for mixing composite liquidcells.

In some embodiments, the network is used for transporting a compositeliquid cell.

In some embodiments, the network comprises a static hydrophobic controlsurface.

In some embodiments, the network comprises a dynamic hydrophobic controlsurface.

In some embodiments, the network comprises a combination of static anddynamic control surfaces.

In some embodiments, real time quantification of proteins is performed.

In some embodiments, the encapsulated sample is dispensed, for example,into a static location.

In some embodiments, biological processing of the encapsulated sample isperformed, and/or the encapsulated sample is combined with one or moreencapsulated samples.

In some embodiments, the system comprises: a conduit for deposition ofencapsulating fluid adjacent to a hydrophobic control surface withperiodic target sample depositions; such that the hydrophobic controlsurface carries the encapsulating fluid with the target sample; ananalysis stage; and a controller to control the system. The depositionmay be continuous.

In some embodiments the invention provides methods and systems forgenerating, and/or transporting, and/or mixing, and/or processingbiological samples. It achieves this by the formation of an immisciblefluid cell within which the biological sample (solid, liquid, emulsionof aqueous-in-oil or suspension of solid in immiscible liquid) may bemanipulated. The method and system can generate, and/or transport,and/or mix, and/or process a range of volumes from micro-litre tosub-nanolitre volumes.

In some embodiments, the invention provides a method and/or a system forprocessing biological samples, comprising encapsulating two or moresamples in a buffer fluid which is immiscible with the samples, toprovide a multi-sample cell, and moving the cell as a combined unit forsample handling.

In some embodiments the method and system may generate non-contaminatingmicrolitre or nanolitre or sub-nanolitre volumes which can be controlledby a number of methods.

In some embodiments it generates one or more biological samples withinan immiscible fluid cell and this composite liquid cell is then placedon a free surface of a mutually immiscible fluid, referred to as acarrier fluid.

The carrier fluid may provide a manipulation platform for the compositeliquid cell. The composite liquid cell may be generated by collectingwithin a tube the composite components in the following sequence:immiscible fluid, biological sample (fluid, emulsion, solid or suspendedparticles), immiscible fluid, and in some cases air. This can berepeated for multiple composite liquid cells with the tube, or in somecases for one or more multi-sample composite liquid cells.

The contents of the tube may then be dispensed on the carrier fluidgenerating the composite liquid cells.

The method and system may generate non-contaminating nanolitre orsub-nanolitre volumes which can be controlled by a number of methods.The invention method provides for the composite liquid cell to becontrolled by electrostatic forces.

In some embodiments the invention may have at least one electricallycharged control surface which can control the composite liquid cell. Theinvention may provide an independent method of control through use ofthe hydrophobic effect.

In some embodiments the invention may have at least one control surfacewhich has a hydrophobicity property which allows adhesion between it andthe outer fluid of an immiscible fluid cell while repelling the internalfluid volume, preventing contamination. Using solid structures embeddedor partly embedded in the carrier oil, the composite liquid cell can beguided in a controlled manner or anchored, increasing the dynamiccontrol of the composite liquid cell.

In some embodiments the invention may provide transport of compositeliquid cells in any of the methods outlined above. Mixing of thecomposite liquid cells in some embodiments involves transporting thecomposite liquid cells to contact, promoting fluid coalescence of theencapsulating oil and biological samples. This mixing processfacilitates the combination of sub-microlitre target volumes with greatefficiency, prevents contamination from other sources and ensures nodead volume per chemistry manipulation. In some embodiments, the samplesare not mixed, resulting in a multi-sample composite liquid cells.

In some embodiments the transport of composite liquid cells through abiological process is not susceptible to air, which leads to theevaporation of target volumes at elevated temperatures. The independenttransport of each composite liquid cell within the system through thecarrier oil reduces power consumption of the overall system that wouldotherwise be required for heating, cooling, or pumping of the carrierfluid, and instead the thermal protocol can be targeted at the compositeliquid cell.

In some embodiments the invention allows for easy automation of thebiochemistry processes. It allows for the dynamic control of individualsamples, thereby allowing for full volume retrieval of samples. Itallows for use in both an open or closed architecture manipulationplatform. It allows for the analysis and manipulation of biologicalsamples. The samples can be analysed easily by optical, acoustic, orelectromagnetic methods.

Individual features of various embodiments disclosed herein may becombined as desired mutatis mutandis, to the extent they are notmutually exclusive to one another.

1-15. (canceled)
 16. A system for producing a composite liquid cell(CLC), the system comprising: a sample-liquid input comprising a sampleliquid; an encapsulating-liquid input comprising an encapsulatingliquid; a carrier liquid; a liquid-handling system; and a controlleroperably connected to the liquid-handling system, wherein the controlleris programmed to cause the liquid-handling system to draw a volume of anencapsulating liquid from the encapsulating-liquid input and a volume ofthe sample liquid from the sample-liquid input and combine the drawnvolumes of encapsulating liquid and sample liquid into an encapsulatedsample on a surface of the carrier liquid to produce a CLC, wherein thesample liquid, the encapsulating liquid and the carrier liquid aremutually immiscible.
 17. The system according to claim 16, wherein thecontroller is programmed to cause the liquid handling system to positionthe volumes of encapsulating liquid and sample liquid onto differentlocations of the surface of the carrier liquid and then combine thepositioned volumes of encapsulating liquid and sample liquid to producea CLC.
 18. The system according to claim 17, wherein the liquid handlingsystem is configured to move the volume of encapsulating liquid acrossthe surface of the carrier liquid.
 19. The system according to claim 16,wherein the controller is programmed to cause the liquid handling systemto combine the volumes of encapsulating liquid and sample liquid into acomposite liquid cell precursor and then deposit the composite liquidcell precursor onto the surface of the carrier liquid to produce a CLC.20. The system according to claim 16, wherein the controller isprogrammed to cause the liquid handling system to position the volume ofencapsulating liquid on the surface of the carrier liquid and thenintroduce the volume of sample liquid into volume of encapsulatingliquid to produce a CLC.
 21. The system according to claim 16, whereinthe liquid-handling system comprises a control tube and a driver. 22.The system according to claim 16, further comprising a motion system.23. The system according to claim 16, wherein the encapsulating liquid,sample liquid and carrier liquid have different densities.
 24. Thesystem according to claim 23, wherein the sample liquid has a densitythat is between the density of the carrier liquid and the density of theencapsulating liquid.
 25. The system according to claim 16, wherein thesample liquid comprises a biological sample.
 26. The system according toclaim 16, wherein the sample liquid comprises an aqueous liquid.
 27. Thesystem according to claim 16, wherein the encapsulating liquid and thecarrier liquid comprise immiscible oils.
 28. A method of producing a CLCon a surface of a carrier liquid, the method comprising: providing avolume of an encapsulating liquid from a encapsulating-liquid input anda volume of the sample liquid from a sample-liquid input; and combiningthe volumes of encapsulating liquid and sample liquid into anencapsulated sample liquid on a surface of a carrier liquid to produce aCLC, wherein the sample liquid, the encapsulating liquid and the carrierliquid are mutually immiscible.
 29. The method according to claim 28,wherein the method comprises positioning the volumes of encapsulatingliquid and sample liquid onto different locations of the surface of thecarrier liquid and then combining the volumes of encapsulating liquidand sample liquid to produce a CLC.
 30. The method according to claim28, wherein the method comprises combining the volumes of encapsulatingliquid and sample liquid into a composite liquid cell precursor and thendepositing the composite liquid cell precursor onto the surface of thecarrier liquid to produce a CLC.
 31. The method according to claim 28,wherein the method comprises positioning the volume of encapsulatingliquid on the surface of the carrier liquid and then introducing thevolume of sample liquid into the volume of encapsulating liquid toproduce a CLC.
 32. The method according to claim 28, wherein the sampleliquid comprises an aqueous liquid and the encapsulating liquid andcarrier liquid comprise immiscible oils.
 33. A system for producing acomposite liquid cell (CLC), the system comprising: a sample-liquidinput; an encapsulating-liquid input; a liquid-handling system; and acontroller operably connected to the liquid-handling system, wherein thecontroller is programmed to cause the liquid-handling system to draw avolume of an encapsulating liquid from the encapsulating-liquid inputand a volume of the sample liquid from the sample-liquid input andcombine the drawn volumes of encapsulating liquid and sample liquid intoan encapsulated sample on a surface of a carrier liquid to produce aCLC, wherein the sample liquid, the encapsulating liquid and the carrierliquid are mutually immiscible.
 34. The system according to claim 33,wherein the controller is programmed to cause the liquid handling systemto position the volumes of encapsulating liquid and sample liquid ontodifferent locations of the surface of a carrier liquid and then combinethe positioned volumes of encapsulating liquid and sample liquid toproduce a CLC.
 35. The system according to claim 16, wherein thecontroller is programmed to cause the liquid handling system to combinethe volumes of encapsulating liquid and sample liquid into a compositeliquid cell precursor and then deposit the composite liquid cellprecursor onto the surface of a carrier liquid to produce a CLC.
 36. Thesystem according to claim 16, wherein the controller is programmed tocause the liquid handling system to position the volume of encapsulatingliquid on the surface of the carrier liquid and then introduce thevolume of sample liquid into volume of encapsulating liquid to produce aCLC.
 37. A device comprising: a composite liquid cell comprising: acarrier liquid; and a sample liquid encapsulated in an encapsulatingliquid on a surface of the carrier liquid; wherein the sample liquid,the encapsulating liquid and the carrier liquid are mutually immiscible.